Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by obstruction of the pulmonary vasculature due (in part) to idiopathic hyper-proliferation of pulmonary arterial smooth muscle cells (PASMC). Preliminary data suggest that this proliferative diathesis relates to excessive mitochondrial fission caused by activation of dynamin related protein 1 (DRP-1). In this proposal we will define the molecular basis for dysregulation of fission in human PAH and explore the nature of the link between a mitochondrial fission-fusion cycle and the cell cycle. We also evaluate whether the requirement of these hyper-proliferative cells for high rates of mitochondrial fission presents an Achilles hee that can be therapeutically targeted. Experiments are performed in human PAH PASMC and lungs and in experimental models (induced by monocrotaline or SU5416 + hypoxia). Preliminary data indicate that human PAH PASMC have fragmented mitochondria, largely due to increased fission. We have developed metrics that use mitochondrial-targeted, photoactivated, green fluorescent protein (mito-paGFP) and 2-photon confocal microscopy to quantify mitochondrial fission. Increased fission in PAH appears to result from phosphorylation of DRP-1 at Serine 616 by the key mitotic regulatory kinase, Cyclin B1- CDK1, thus linking fission and mitosis. Elevated cytosolic calcium in PAH also promotes DRP-1-mediated fission by: a) activation of calmodulin kinase and b) calcineurin-mediated dephosphorylation of an inhibitory form of DRP- 1 (Phos-Ser637). Thus, increased activity of calcineurin, cyclin B1-CDK1 and DRP-1 promote fission and proliferation in human PAH PASMC; conversely, DRP-1 inhibition (via the small molecule inhibitor, mdivi-1, or siDRP-1) decreases PASMC proliferation and, in the case of mdivi-1, regresses PAH in vivo. Hypothesis: Pathologic DRP-1 activation increases fission and promotes excessive PASMC proliferation in PAH. Corollary: DRP-1 inhibition prevents cell cycle progression, causing G2-M phase arrest. Anti-fission therapies may constitute an antiproliferative therapy for PAH. Aim 1) Are DRP-1 activation and fission required for the hyperproliferation of PASMC in human PAH? Aim 2) Does post-translational DRP-1 modification regulate PASMC proliferation in PAH? Aim 3) Does inhibition of mitochondrial fission have therapeutic benefit in experimental PAH? Innovation and Impact: This proposal is innovative in recognizing that mitochondrial checkpoints, related to DRP-1 activation and fission, regulate cell cycle progression. The concept that mitochondrial fission is tightly linked to cell cycle progression via shared regulatory kinases is also novel. The discovery that inhibiting DRP-1 (directly or by targeting its regulatory kinases) is anti-proliferative offers a new, anti-fission profusion, therapeutic paradigm for PAH. The translational potential of this proposal is enhanced by careful correlation of results in rodent PAH models with findings in human PAH lungs/cells from a well-characterized cohort. This is the first study to exploit PAH's reliance on rapid fission to devise novel anti-fission, anti-proliferative therapies for PAH.